Selection for increased reproductive rate (fitness) requires sustained growth, which is achieved through regular dilution of a growing culture. In the prior art this has been accomplished two ways: serial dilution and continuous culture, which differ primarily in the degree of dilution.
Serial culture involves repetitive transfer of a small volume of grown culture to a much larger vessel containing fresh growth medium. When the cultured cells have grown to saturation in the new vessel, the process is repeated. This method has been used to achieve the longest demonstrations of sustained culture in the literature (Lenski & Travisano: Dynamics of adaptation and diversification: a 10,000-generation experiment with bacterial populations. 1994. Proc Natl Acad Sci USA. 15:6808-14), in experiments which clearly demonstrated consistent improvement in reproductive rate over period of years. This process is usually done manually, with considerable labor investment, and is subject to contamination through exposure to the outside environment. Serial culture is also inefficient, as described in the following paragraph.
The rate of selection, or the rate of improvement in reproductive rate, is dependant on population size (Fisher: The Genetical Theory of Natural Selection. 1930. Oxford University Press, London, UK). Furthermore, in a situation like serial transfer where population size fluctuates rapidly, selection is proportional to the harmonic mean (Ñ) of the population (Wright: Size of population and breeding structure in relation to evolution. 1938. Science 87: 430-431), and hence can be approximated by the lowest population during the cycle.
Population size can be sustained, and selection therefore made more efficient, through continuous culture. Continuous culture, as distinguished from serial dilution, involves smaller relative volume such that a small portion of a growing culture is regularly replaced by an equal volume of fresh growth medium. This process maximizes the effective population size by increasing its minimum size during cyclical dilution. Devices allowing continuous culture are termed “chemostats” if dilutions occur at specified time intervals, and “turbidostats” if dilution occur automatically when the culture grows to a specific density.
For the sake of simplicity, both types of devices will hereafter be grouped under the term “chemostat”. Chemostats were invented simultaneously by two groups in the 1950's (Novick & Szilard: Description of the chemostat. 1950. Science 112: 715-716) and (Monod: La technique de la culture continue—Théorie et applications. 1950. Ann. Inst. Pasteur 79:390-410). Chemostats have been used to demonstrate short periods of rapid improvement in reproductive rate (Dykhuizen D E. Chemostats used for studying natural selection and adaptive evolution. 1993. Methods Enzymol. 224:613-31).
Traditional chemostats are unable to sustain long periods of selection for increased reproduction rate, due to the unintended selection of dilution-resistant (static) variants. These variants are able to resist dilution by adhering to the surface of the chemostat, and by doing so, outcompete less sticky individuals including those that have higher reproductive rates, thus obviating the intended purpose of the device (Chao & Ramsdell: The effects of wall populations on coexistence of bacteria in the liquid phase of chemostat cultures, 1985. J. Gen. Microbiol. 131: 1229-36).
One method and chemostatic device (the Genetic Engine) has been invented to avoid dilution resistance in continuous culture (U.S. Pat. No. 6,686,194-B1 filed by PASTEUR INSTITUT [FR] & MUTZEL RUPERT [DE]). This method uses valve controlled fluid transfer to periodically move the growing culture between two chemostats, allowing each to be sterilized and rinsed between periods of active culture growth. The regular sterilization cycles prevent selection of dilution-resistant variants by destroying them. This method and device achieves the goal, but requires independent complex manipulations of several fluids within a sterile (sealed) environment, including one (NaOH) which is both very caustic and potentially very reactive, quickly damaging valves, and posing containment and waste-disposal problems. The chemostatic device is also limited in that no provisions are made to provide a support for cells to grow on There are some types of cells that are difficult to culture in large amounts due to the conditions the cells require to survive and grow. It is believed that these cells could grow in conditions provided by a continuous culture approach. This is particularly the case for stem cells.
For example, human embryonic stem cells are typically grown by isolating and transferring a stem cell mass into a plastic laboratory culture dish that contains a nutrient broth known as culture medium. The cells divide and spread over the surface of the dish. The inner surface of the culture dish is typically coated with mouse embryonic skin cells that have been treated so they will not divide. This coating layer of cells is called a feeder layer. The reason for having the feeder layer in the bottom of the culture dish is to give the human embryonic stem cells a sticky surface to which they can attach. Also, the feeder cells release nutrients into the culture medium. Recently, scientists have begun to devise ways of growing embryonic stem cells without the mouse feeder cells. This is a significant scientific advancement because it avoids the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human cells.
Over the course of several days, the cells of the inner cell mass proliferate and begin to crowd the culture dish. When this occurs, they are removed gently and plated into several fresh culture dishes. The process of replating the cells is repeated many times and for many months, and is called subculturing. Each cycle of subculturing the cells is referred to as a passage. After six months or more, the original cells of the cell mass yield millions of embryonic stem cells. Embryonic stem cells that have proliferated in cell culture for six or more months without differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic stem cell line.
Once cell lines are established, or even before that stage, batches of them can be frozen and shipped to other laboratories for further culture and experimentation. However, continuous culture grants the advantage of suppressing a maximum of manipulations that stress the living cells and create a potential source of contamination. When a culture is started, continuous culture conditions allow the skilled artisan to take advantage of a continuous production of cells. Once stem cells are being produced, the production of stems cells could continue without interruption to produce substantially more stem cells than methods that are typically used today.